JPH075161A - Filler for high performance liquid chromatography - Google Patents

Abstract

PURPOSE:To obtain a filler for reversed phase distribution chromatography for identifying the molecular profile without using the nonpolar aromaticity by a constitution wherein a copolymer, which can be represented by a chemical structural formula, is bonded to a silica gel through terminal X. CONSTITUTION:The filter for reverse phase distribution chromatography has no aromaticity and accomplishes separation by carrying the nonpolarity of high molecular orientation on a silica gel. Consequently, a reversed phase chromatography for identifying the molecular profile through the use of molecular orientation of nonpolar phase is obtained. Since the nonpolar phase being carried has no aromaticity, undesired pi-pi interaction with salute molecule does not take place. Furthermore, since the nonpolar phase to be carried is a copolymer having a functional group X at one end thereof coupled with the silica gel, flexibility is provided and the extent of molecular orientation varies with the temperature. The holding time of solute can be controlled according to the variation of molecular orientation and the temperature is regulated to shorten the time required for analysis.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel packing material for reversed phase partition chromatography.

[0002]

2. Description of the Related Art Liquid chromatography has been widely used in the analysis and separation of organic compounds, pharmaceuticals, foods and the like. In particular, reversed-phase partition chromatography has been attracting attention as an excellent method because of its wide range of target compounds and simple separation mechanism. As a packing material for conventional reversed phase partition chromatography,
For example, a filler in which the surface of silica gel is made nonpolar by an octyl group or an octadecyl group is known.

[0003]

However, in the packing material such as the silica gel particles, the separation is carried out only by utilizing the difference in polarity between the non-polar phase and the elution phase introduced on the silica gel. However, when the polarities of the substances to be separated are similar to each other, there is a drawback that good separation cannot be achieved. Also, when the polarity of the substances to be separated is significantly different or low, excessive retention of the filler is observed,
In this case, a special separation operation called gradient elution is required in order to shorten the analysis time, and there is also a problem that the operation becomes complicated.

On the other hand, as a method for depolarizing silica gel, an example in which an aromatic rigid molecule such as a biphenyl group is introduced has been reported. In this case, not only the separation is caused by the difference in polarity but also the molecular It is performed by identifying differences in aromaticity and shape. However, it is generally considered that the aromaticity of the packing material causes peak tailing in chromatography and is not preferable. For example, styrene-divinylbenzene copolymer particles with high aromaticity are known as packing materials for liquid chromatography, and although they are used as packing materials for ion exchangers and gel permeation chromatography, they have reversed phase partitioning. Its use as a packing material for chromatography is very limited. this is,
This is because the peak tailing based on the π-π interaction between the aromaticity of the styrene-divinylbenzene copolymer and the π-electrons of the solute is often a problem.

It is an object of the present invention to provide a packing material for reversed phase partition chromatography that distinguishes the shape of a molecule without using an aromatic non-polar phase. This is achieved because the non-polar phase supported on silica gel has a molecular orientation. Another object of the present invention is to provide a filler in which the supported non-polar phase is a polymer and one end of the polymer is bonded to silica gel.

[0006]

According to the present invention, the general formula (Formula 1)

(In the formula, R ₁ represents a hydrogen atom or a methyl group.
R ₂ contains at least (CH ₂ ) _m CH ₃ , and m is ₃ to _2.
1, preferably 8 to 21. n represents 2-200, preferably 5-50. The polymer represented by) is bonded to silica gel via the terminal X to provide a packing material for reverse phase partition chromatography.

The present invention will be described in more detail below. The packing material for reversed phase partition chromatography of the present invention is a packing material obtained by binding a polymer represented by Chemical formula 1 to silica gel via a terminal X.

R _{1 in} Chemical formula ₁ is a hydrogen atom or a methyl group. R ₂ is a substituent containing an alkyl group having 4 to 22 carbon atoms. When the number of carbon atoms of R ₂ is less than 4, the hydrophobicity is too small and the retention capacity as a packing material for reverse phase partition chromatography becomes small, resulting in poor separation performance, which is not preferable. In addition, the R ₂ is the larger number of carbon atoms, R ₂
The molecular orientation between them becomes high, and as a result, the separation ability also becomes high, but it is difficult to obtain the raw materials for the compounds having 23 or more carbon atoms,
There is a problem that it is difficult to obtain a polymer because the melting point is too high, and it is not practical. R represented by the general formula (Formula 1)
Examples of ₂ include butyloxy group, hexyloxy group, octyloxy group, decyloxy group, dodecyloxy group, tetradecyloxy group, hexadecyloxy group,
Ester residues of long chain alcohols such as octadecyloxy group and long chain amines such as butylamino group, hexylamino group, octylamino group, decylamino group, dodecylamino group, tetradecylamino group, hexadecylamino group, octadecylamino group Preferable examples thereof include amide residues and the like, and when used, they can be used alone or as a mixture.

X in the chemical formula 1 is a functional group for bonding the polymer represented by the chemical formula 1 to silica gel. For example, a propyltrimethoxysilyl group which directly reacts with the silica gel and bonds thereto is preferable. . However, when the silica gel is previously aminated, carboxylated, or hydroxylated, a substituent containing a carboxyl group, a hydroxyl group, or an amino group can be used for X, and in this case, a substituent such as dicyclohexylcarbodiimide can be used. The polymer represented by Chemical formula 1 can be bonded to silica gel using a condensing agent. Y in Chemical formula 1 may be any residue as long as it does not particularly affect the chromatographic properties, and a hydrogen atom is preferred.

In the present invention, the polymer represented by the chemical formula 1 is most conveniently the monomer represented by the chemical formula 2 satisfying the conditions of R ₁ and R ₂ is taxogen and the terogen represented by the chemical formula 3 is used. It is obtained by the telomerization method.

[0011]

Embedded image CH ₂ ═C (R ₁ ) CO—R ₂

[0012]

[Image Omitted] X- (CH ₂ ) _p -Y

The taxogen represented by Chemical Formula 2 satisfying the conditions of R ₁ and R ₂ is butyl acrylate, butyl methacrylate, butyl acrylamide, butyl methacrylamide, hexyl acrylate, hexyl methacrylate, hexyl acrylamide, hexyl methacrylamide, octyl. Acrylate, octyl methacrylate, octyl acrylamide, octyl methacrylamide, decyl acrylate, decyl methacrylate, decyl acrylamide, decyl methacrylamide, dodecyl acrylate, dodecyl methacrylate, dodecyl acrylamide, dodecyl methacrylamide, tetradecyl acrylate, tetradecyl methacrylate, tetradecyl acrylamide, Tetradecyl methacrylamide,
Preferable examples include hexadecyl acrylate, hexadecyl methacrylate, hexadecyl acrylamide, hexadecyl methacrylamide, octadecyl acrylate, octadecyl methacrylate, octadecyl acrylamide, octadecyl methacrylamide. Chemical 3
As the telogen represented by, X is a functional group capable of reacting directly with silica gel and binding thereto, and Y is a functional group having a high chain transfer constant. Although the alkyl chain length (p) of the spacer which connects X and Y is not particularly limited,
Those having p of 2 to 6 can be synthesized. 3-mercaptopropyltrimethoxysilane is preferably mentioned as a telogen represented by Chemical formula 3 which satisfies such conditions, but 3 is preferable for particles obtained by preamination of silica gel.
Examples include -mercaptobutanoic acid and 5-mercaptohexanoic acid, and for particles obtained by pre-carboxylating or hydroxylating silica gel, 3-mercaptopropylamine, 6-mercaptohexylamine, and the like.

In the present invention, the polymerization degree of the polymer represented by Chemical Formula 1 must be at least 2 to 200. A polymerization degree of 5 to 50 is suitable, and a polymerization degree of 5 to 5 is preferable.
In the case of the polymer of 0, the side chain R ₂ is well molecularly oriented, and as a result, good separability is obtained. A polymer having a degree of polymerization of 51 to 200 also has a good molecular orientation, but the ratio of the residue X in the chemical formula 1 in one molecule decreases, and the reactivity with silica gel deteriorates. In the case of a polymer having a degree of polymerization of 201 or more, not only the proportion of the residue X is remarkably reduced but also the solubility is reduced, so that it is practically impossible to bond to silica gel. The degree of polymerization can be easily adjusted by adjusting the mixing molar ratio of terogen and taxogen when using a telogen having a mercapto group having a high chain transfer constant such as 3-mercaptopropyltrimethoxysilane.

In the present invention, there are roughly two methods for supporting the polymer represented by Chemical formula 1 on the silica gel particles. When the polymer X represented by Chemical formula 1 can directly react with the silanol residue of the silica gel like a trimethoxysilyl group, the polymer supported on silica gel is mixed in an organic solvent and gently stirred. While at 60-80 ℃ 1
The polymer can be bonded to silica gel by keeping it for about 2 hours. As the organic solvent, carbon tetrachloride,
Chloroform, tetrahydrofuran, dioxane and the like can be used. On the other hand, when the polymer X represented by Chemical formula 1 contains a carboxyl group, partially aminated silica gel is used, and when the polymer X represented by Chemical formula 1 contains an amino group or a hydroxyl group, The partially carboxylated silica gel can be used to attach the polymer to the silica gel by conventional condensation methods. As the condensing agent, dicyclohexylcarbodiimide, diethyl phosphoric acid cyanide, or the like can be used.

In the packing material for reversed phase partition chromatography of the present invention, in order to improve the separation performance, the silica gel serving as a carrier is preferably porous spherical particles. in this case,
The sphere diameter strongly depends on the purpose of separation, but in the case of use mainly for analysis, the diameter is several μm to 10 μm, and the one having a narrow particle size distribution is used. On the other hand, in the case of the use mainly for high-speed large-volume fractionation, particles having a larger particle size, for example, several tens, are taken into consideration in consideration of the high flow rate characteristic in chromatography.
The one with μm is used.

The packing material for reversed phase partition chromatography of the present invention can be used by a method of packing it in a column, like the usual silica gel packing material for reversed phase partition chromatography.

[0018]

EFFECTS OF THE INVENTION The packing material for reversed phase partition chromatography of the present invention has a highly molecularly oriented non-polar phase having no aromaticity and is supported on silica gel. Reversed phase partition chromatography can be provided that achieves separations similar to those of a chromatographic packing material, while discriminating the shape of the molecule by utilizing the molecular orientation of the non-polar phase. Furthermore, the supported non-polar phase does not exhibit aromaticity and therefore does not exhibit undesired π-π interactions with solute molecules. Furthermore, the supported non-polar phase is a polymer, and since one end of the polymer is bonded to silica gel, it has flexibility, and therefore exhibits a change in the degree of molecular orientation with temperature. This change in the degree of molecular orientation can be used to control the retention time of the solute. Therefore, even in the case of solutes that exhibit excessive retention, the temperature can be adjusted without using a special gradient elution method. The analysis time can be shortened.

[0019]

EXAMPLES The present invention will be described in more detail with reference to Examples, Test Examples and Comparative Examples, but the present invention is not limited to these.

Example 1 Ruacrylate (Taxogen) and 3-mercaptopropyltrimethoxysilane (Terogen) were put together with ethanol and stirred for 10 minutes while introducing nitrogen gas. Then, azobisisobutyronitrile was added as an initiator, and radical telomerization was carried out under the nitrogen gas atmosphere at 80 ° C. for 6 hours at the charging ratio of terogen and taxogen shown in Table 1. The mixture was allowed to cool to room temperature, the precipitate was filtered off on a G-5 glass filter, washed thoroughly with methanol and acetone, and dried under reduced pressure to obtain the desired polymer. Hereinafter, the same polymer is abbreviated as ODA _n . In this abbreviation, n represents the average degree of polymerization.

[0021]

[Table 1]

The obtained ODA _n was dissolved in deuterated chloroform and the average degree of polymerization n was determined by H-NMR. Also,
The phase transition temperature of ODA _n was determined by differential scanning calorimetry. The obtained ODA _n and silica gel (dry state) were put in a three-neck round bottom flask together with carbon tetrachloride in the mixing ratio shown in Table 2. Attach a stirring seal and a reflux condenser to this, and
The mixture was placed in an oil bath at 0 ° C. and stirred for 12 hours. After stirring, G
5 After collecting with a glass filter and washing with carbon tetrachloride, silica gel carrying ODA _n was obtained by filtration. The supported amount was determined by elemental analysis, and the structure of the supported substance was confirmed by infrared absorption spectrum and differential scanning calorimetry analysis.

[0023]

[Table 2]

Examples 2 to 6 The desired polymer was obtained in the same manner as in Example 1 except that the type of taxogen, the charging ratio thereof, the solvent, the reaction temperature and the like were changed as shown in Table 1. The abbreviations for the polymers were prepared according to the type of taxogen used and are shown in Table 1.
In this abbreviation, n represents the average degree of polymerization. The loading of the polymer on silica gel was carried out in the same manner as in Example 1 by loading the polymer on silica gel.

Example 7 The desired polymer was obtained in the same manner as in Example 1 except that the types of telogen, the charging ratio thereof, the solvent, the reaction temperature and the like were changed as shown in Table 3. The abbreviations for the polymers were prepared according to the type of telogen used and are shown in Table 3. still,
In this abbreviation, n represents the average degree of polymerization.

[0026]

[Table 3]

The obtained polymer and partially aminated silica gel (in a dry state) were placed in a three-necked round bottom flask together with tetrahydrofuran in a mixing ratio shown in Table 2. Didichlorohexylcarbodiimide was added thereto, and the mixture was equipped with a stirring seal and a reflux condenser and stirred at room temperature for 24 hours. After stirring, G5
After collecting with a glass filter and washing with tetrahydrofuran, silica gel carrying a polymer was obtained by filtration. The supported amount was determined by elemental analysis, and the structure of the supported substance was confirmed by infrared absorption spectrum and differential scanning calorimetry analysis.

Examples 8 to 10 The desired polymer was obtained in the same manner as in Example 1 except that the types of telogen, the charging ratio thereof, the solvent, the reaction temperature and the like were changed as shown in Table 3. The abbreviations of the polymers were prepared according to the type of telogen used and are shown in Table 3. still,
In this abbreviation, n represents the average degree of polymerization. Loading of the polymer on silica gel was changed except that the mixing ratio of the polymer and silica gel, the reaction solvent, the temperature, etc. were changed as shown in Table 2.
The polymer was supported on silica gel in the same manner as in Example 7.

Test Example 1 3.3 g of particles D (dry state), 1-hexanol 15
ml and chloroform 15 ml were placed in a 100 ml beaker and dispersed using an ultrasonic cleaner for about 15 minutes. This was placed in a packer connected to a stainless steel column having a diameter of 4.6 mm and a length of 30 cm, the void was filled with a mixed solvent of 1-hexanol and chloroform (1: 1), and then chloroform was flown at a flow rate of 8 ml / min. did. Pressure is 450
The flow rate was adjusted so as to be kg / cm ² , and loroform was further flowed at the same pressure for 30 minutes. Remove the column from the packer, attach a filter, and 100 m at a flow rate of 1 ml / min.
The filling was completed by flushing with 1 l of chloroform and 100 ml of methanol. Liquid chromatography was carried out using benzene, ethylbenzene, butylbenzene, hexylbenzene, and octylbenzene as samples, and methanol / water (9: 1) as the eluent. As shown in FIG. 1, a chromatogram in which all samples were completely separated was obtained.

Test Example 2 After packing particles A in the same manner as in Test Example 1,
Liquid chromatography was performed using non-planar o-terphenyl and planar triphenylene as samples and methanol as the eluent. As shown in FIG.
A chromatogram was obtained in which the two samples were completely separated. The α value, which is a measure of separability, was 4.5.

Test Example 3 With respect to the column prepared in Test Example 2, liquid chromatography was carried out using benzene, naphthalene, anthracene, pyrene and triphenylene as polycyclic aromatic compounds as a sample and methanol as an eluent. As shown in FIG. 3, it was confirmed that there was no unfavorable peak tailing and that all samples could be completely separated.

Test Example 4 With respect to the column prepared in Test Example 2, liquid chromatography was carried out using anthracene and triphenylene as samples and methanol as an eluent at different temperatures. As a result, a graph (FIG. 4) showing a special temperature dependence with respect to the holding time was obtained. In addition, it was confirmed that the temperature at which the retention time significantly changes coincides with the phase transition temperature of the supported nonpolar phase.

Comparative Example 1 OD as a commercially available packing material for reversed phase partition chromatography
Using S, the same examination as in Test Example 4 was performed. Separability α value of o-terphenyl and triphenylene is only 1.4
Met.

Comparative Example 2 OD as a packing material for commercially available reverse phase partition chromatography
Using S, the same examination as in Test Example 3 was performed. A graph (FIG. 5) showing a linear temperature dependence with respect to the retention time was obtained. It was confirmed by differential scanning calorimetry that the ODS used did not show any phase transition behavior within the measurement temperature range.

[Brief description of drawings]

FIG. 1 shows a chromatogram in Test Example 1 in which the separation of a mixture of benzene, ethylbenzene, butylbenzene, hexylbenzene, and octylbenzene was tested using particles D.

FIG. 2 shows a chromatogram in Test Example 2 in which the separation of a mixture of o-terphenyl and triphenylene was tested using Particle A.

FIG. 3 shows a chromatogram in Test Example 3 in which the separation of a mixture of benzene, naphthalene, anthracene, pyrene and triphenylene was tested using Particle A.

FIG. 4 shows the results of plotting the retention capacities of particles A for anthracene and triphenylene against temperature in Test Example 4. T _c in the figure represents the phase transition temperature of the particle A.

FIG. 5 shows the results of plotting the retention capacity of ODS for anthracene and triphenylene in Comparative Example 2 against temperature.

Claims (4)

[Claims] 1. A silica gel particle in which a polymer represented by the general formula (Formula 1) is bonded via a terminal X. [Chemical 1] 2. The particle according to claim 1, wherein n in Chemical formula 1 is 2 to 200. 3. The particle according to claim 1, wherein R ₂ in Chemical formula 1 contains at least a (CH ₂ ) _m CH ₃ group in the chemical structure, and m is ₃ to 21. 4. The particle according to claim 1, wherein R _{1 in} Chemical formula ₁ is a hydrogen atom or a methyl group.